One of the major functions of the renal proximal tubule is to secrete acid into the tubule lumen, thereby reabsorbing most of the filtered HCO-3, and exporting the acid load produced by metabolism throughout the body. Although it is well established that the proximal tubule can modulate its rate of acid secretion in response to changes in the acid base status of the blood, the mechanisms by which this occurs over ra short time frame are not at all clear. The long-term goal of this project is to understand how changes in blood [C02], [HCO-3] and pH acutely control HCO-3 reabsorption by the proximal tubule. The general approach for the proposed experiments will be isolated and perfuse in vitro single proximal tissues isolated from the kidney of the rabbit. In some experiments, the combination of microcalorimetry and 3/H-inulin will be used to measure HCO-3 reabsorption (JHCO3) and volume reabsorption (Jv). In other experiments, digital imaging of fluorescence signals will be used to monitor intracellular pH (Ph/I) or calcium ([Ca/++]i) in tubule cells. A novel aspect of the proposed experiments is the possibility of switching the basolateral ("bath) solution to an out-of-equilibrium (OOE) CO2/HCO-3 solution with virtually any combination of [CO2], [HCO-3] and pH. Preliminary data on tubules perfused with CO2/HCO-3 suggest that switching the bath solution from CO2/HCO-3 free HEPES to an OOE "pure HCO-3" solution (containing a physiological [HCO-3] and pH, but negligible CO2) markedly reduces JHCO3 and JV. Conversely, "pure CO2" (physiological [CO2] and pH, but negligible NCO-3) in the bath markedly increases JHCO/3 and JV. Switching the bath to CO2/HCO-3 also causes a substantial pH/i increase, due to stimulation of apical Na-H exchange and H/+ pumping. The proposed work has four aims: First, how do isolated changes in [HCO- 3]BL or pH/BL affect JHCO/3 and Jv? The approach will be either to switch the bath from HEPES to a "pure HCO-3" solution of varying basolateral [HCO-3] (i.e., [HCO-3]BL), or from a standard CO2-HCO-3 solution to an OOE solution in which only [HCO-3]BL has been changed. In other experiments, pH/BL will be varied, rather than [HCO-3]BL. Second, how do isolated changes in [HCO-3]BL or pH/BL affect the HCO-3 efflux mediated by the electrogenic Na/HCO3 co-transporter (NBC)? The approach will be express NBC in Xenopus oocytes, and study the sensitivity of NBC to isolated changes in either [HCO-3]0 PR Ph/0. The goal is to determine the extent to which the data obtained in Aim 1 can be accounted by for effects on NBC. Third, how do isolated changes in [CO2]/BL affect JHCO3 and JV? The approach will be the same as in Aim 1, except that [CO2]BL (rather than [HCO-3]BL or pH/BL) will be changed. Fourth, how does the PT cell transduce an increase in [C02]BL to an increase in H/+ secretion? The approach will be to determine if changes in [Ca/++]i, nitric oxide production, protein phosphorylation or insertion of membrane vesicles contribute to the changes in JHC03 observed in Aim 3. The proposed work should provide fresh insight into how blood acid-base parameters acutely regulate HCO-3 reabsorption by the proximal tubule.